In recent years, N-acetylneuraminic acid is noted as raw materials of drugs. It is known that said N-acetylneuraminic acid may be enzymatically synthesized from N-acetylmannosamine and pyruvic acid using N-acetylneuraminic acid lyase. However, because of expensiveness and difficulty of large-scale preparation of N-acetylmannosamine, a method for preparing N-acetylneuraminic acid by reacting inexpensive N-acetylglucosamine and pyruvic acid in the presence of N-acetylneuraminic acid lyase is proposed (Udo Kragl et al., Angewandte Chemi-International Edition in English, 30, 827-828 (1991)). This method utilizes that acylglucosamine 2-epimerase epimerizes N-acetyl-glucosamine to N-acetylmannosamine. However, acylglucosamine 2-epimerase employed in this method exists only in a trace amount in animal tissues and techniques of large-scale production thereof has not been developed. Accordingly, above-mentioned method may not be employed practically.
On the other hand, Teshima et al. (Clinical Chemistry, 34, 2291-2294 (1988)) disclose that acylglucosamine 2-epimerase is useful for determination of N-acetylhexosamine.
As shown above, acylglucosamine 2-epimerase is a very important enzyme and establishment of an efficient method for production thereof is earnestly desired.
It is known that acylglucosamine 2-epimerase exists in animal tissues. For example, Asis Datta (Methods in Enzymology, 41, 407-412 (1975)) reported that acylglucosamine 2-epimerase existed in porcine kidney. It also widely exists in kidney, liver, mucosal cell, submandibular gland, intestinal mucosa, colon, salivary gland, etc.
Purification of acylglucosamine 2-epimerase from animal tissues is, however, very difficult, and only crude acylglucosamine 2-epimerase is obtained up to the present. For example, Ghosh et al (Methods in Enzymology, 8, 191-195 (1966)) and Asis Datta (Methods in Enzymology, 41, 407-412 (1975)) tried to isolate and purify acylglucosamine 2-epimerase. However, degree of purity is low according to the report of Ghosh. According to Asis Datta, specific activity thereof is about as low as 6 unit/mg protein.
These reports demonstrate that purification of enzyme from crude extract of porcine kidney cortex prepared by homogenizer followed by a combination of conventional purification means, such as protamine concentration, bentonite treatment, DEAE-cellulose column chromatography, adsorption on calcium phosphate gel, etc. is difficult.
The inventors further conduct gel filtration, hydroxyapatite, hydrophobic gel and like a variety of chromatographies and chromatofocusing in addition to said purification means, which do not lead to recovering said enzyme in a purified form due to dilution of enzymatic activities and loss of enzymatic activities caused by inactivation of the enzyme. A trace amount of existence of the enzyme in kidney is one of reasons for difficulties of purification thereof.
Recently, preparation of heterologous proteins using microorganisms becomes relatively easy with the progress of gene recombination techniques. However, because of necessity of isolation of protein for utilizing said means, materials to specify said enzyme, such as DNA probes and antibodies may not be prepared with respect to acylglucosamine 2-epimerase which is obtained only in a crude form. In that case, a conventional alternative method comprises electrophoresis of a partially purified enzyme on polyacrylamide gel, blotting the enzyme on polyvinylidenedifluoride (PVDF) membrane to analyze amino acid sequence thereof, synthesizing DNA probes based on said amino acid sequence to detect a desired gene. However, with respect to this enzyme, the amino acid sequence may not be determined by said method, because N-terminal of acylglucosamine 2-epimerase is blocked by an unknown residue.
As shown above, any method generally employed as gene recombination techniques may not be applicable to acylglucosamine 2-epimerase. A way of producing this enzyme by gene recombination techniques has been closed.
It is an object of the invention to provide a method for producing acylglucosamine 2-epimerase in large quantities at low cost.
In addition, it is another object of the invention to provide acylglucosamine 2-epimerase.
Further, it is another object of the invention to provide DNA molecules coding for acylglucosamine 2-epimerase.
Furthermore, it is another object of the invention to provide recombinant vectors into which a DNA molecule coding for acylglucosamine 2-epimerase is integrated.
Furthermore, it is another object of the invention to provide transformants, wherein recombinant vectors into which a DNA molecule coding for acylglucosamine 2-epimerase is integrated is introduced.
Furthermore, it is another object of the invention to provide antihypertensive agents.
Furthermore, it is another object of the invention to provide epimerizing agents converting N-acetylglucosamine to N-acetylmannosamine.
Furthermore, it is another object of the invention to provide methods for producing N-acetylmannosamine.
Furthermore, it is another object of the invention to provide methods for producing N-acetylneuraminic acid.
Furthermore, it is another object of the invention to provide novel polypeptides.